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Cloning from cDNA - the wrong gene?!


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#1 WonderRakeWoman

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Posted 06 April 2010 - 11:50 AM

Hello cloning-type people,

I am trying to clone my gene of interest into a GFP vector. I made cDNA from RNA, used gene-specific primers (with overhangs for restriction digest) for PCR, isolated a band of roughly the expected size, digested both the vector and the insert and ligated. I sequenced plasmid DNA from the colonies I got, and a lot of them seem to have a piece of a random other gene inserted into the MCS.

I noticed that there was something inserted in the MCS that did not match my vector or gene of interest so I did a Blast search and got a partial match with another gene's coding DNA. I did a digest map of this new DNA and my 2 restriction enzymes cut out a 200 base piece of this new DNA. When comparing this piece that would be cut out with my 2 enzymes to the sequences of my plasmids, it's an exact match! So it looks as though I somehow amplified the wrong gene and inserted a piece of it into my vector.

I just sent the empty vector over to be sequenced today to make sure that this insert didn't come from the vector originally, but I am stumped as to how this could have happened. Doing a Blast search with my PCR primers showed no sequence similarity to this random gene that appears to have been amplified. Anyone have any idea how this could happen?

Thanks!

#2 pcrman

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Posted 06 April 2010 - 07:23 PM

It is not that unusal amplying and cloning unrelated sequences even the size of PCR product is right.

Here are my suggestions:

If you have additional clones, first verify them by restriction digestion using an enzyme that cuts your insert and then sequence ones that give the right fragments.

You can also digest your PCR product before ligation to make sure the amplification is specific. Make sure your pcr product is well separated by agarose gel to get rid of any non specific bands

Are the sequencing primers on the vector or insert? If on the insert, you should use primers that bind vector sequence.

#3 WonderRakeWoman

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Posted 07 April 2010 - 07:02 AM

It is not that unusal amplying and cloning unrelated sequences even the size of PCR product is right.

Here are my suggestions:

If you have additional clones, first verify them by restriction digestion using an enzyme that cuts your insert and then sequence ones that give the right fragments.

You can also digest your PCR product before ligation to make sure the amplification is specific. Make sure your pcr product is well separated by agarose gel to get rid of any non specific bands

Are the sequencing primers on the vector or insert? If on the insert, you should use primers that bind vector sequence.



I looked at all of the clones that I got, and they all either have no insert or a random insert (there were 2 wtih one specific piece of DNA inserted and 3 with another). My sequencing primers are on the vector, next to the MCS (I had to design them myself, so that's not an issue for me). I didn't have much insert DNA to work with after the ligation, so I didn't run it on a gel - next time I definitely will! Thanks for the suggestions.




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